T cells are the major cellular arm of the immune system that elicit potent and specific immune responses in vivo against bacterial and viral antigens. Individuals born with severe combined immunodeficiency (SCID) exhibit a complete absence of T cells, while individuals infected with HIV/AIDS or treated for cancer with chemo/radio-therapy exhibit a profound depletion of T cells. Regardless of whether the immunodeficiency is congenital or acquired, these individuals are compromised in their capacity to generate new T cells from incoming bone marrow-derived stem cells and to mount sufficient immune responses against opportunistic infections. Conversely, individuals with certain autoimmune diseases such as arthritis and diabetes exhibit inappropriate immune responses against self-tissue due in part to an absence of a particular kind of T cell termed T-regulatory cells. Thus, the ability to generate new designer T cells in vitro through the differentiation of expanded progenitor cells extracted from a particular individual may offer therapeutic benefits in the treatment of many diseases by restoring T cell numbers and the capacity to maintain and regulate a functional immune system. An in vitro differentiation system in which mouse hematopoietic stem cells are induced to differentiate towards a T cell lineage following a period of coculture with the mouse OP9 bone marrow stromal cell line that expresses the Notch receptor ligands Delta-like-1 or -4 has been described, however, the characterization human hematopoietic stem cells using the same system has yet to be elucidated.
Hematopoietic stem cells (HSCs) which give rise to erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage specific markers (termed Lin−) (Kawamoto et al., 1997). Human umbilical cord blood (CB) provides a rich source of HSCs, which are comparable to bone marrow-derived HSCs (Barker and Wagner, 2003; de Wynter et al., 1999; Fisher et al., 1990; Galy et al., 1993; Gluckman et al., 1997; Ito et al., 2002; Lewis and Verfaillie, 2000; McCune et al., 1991; Sánchez et al., 1993; Wilpshaar et al., 2002). Human T cells differentiate in the thymus via discrete developmentally-regulated steps that involve a series of commitment events and developmental checkpoints including T cell receptor (TCR) variable (V), diversity (D), and joining (J) gene segment rearrangements [V(D)J], and positive/negative selection of developing thymocytes (Spits, 2002). The earliest intrathymic progenitors express high levels of CD34 and CD7, do not express CD1a, and are triple-negative (TN) for mature T cell markers: CD4, CD8, and CD3 (Galy et al., 1993). Commitment to the T cell lineage is associated with the expression of CD1a by CD7-expressing pro-thymocytes (Spits, 2002; Spits et al., 2000).
Several studies have implicated the Notch pathway in promoting HSC expansion, self-renewal (Stier et al., 2002), survival (Deftos and Bevan, 2000; Osborne and Miele, 1999), and the induction of T cell lineage commitment (MacDonald et al., 2001; Osborne and Miele, 1999; Pear and Radtke, 2003; Radtke et al., 2002; Robey, 1999; von Boehmer, 2001). In humans there are four Notch receptors (Ellisen et al., 1991; Lardelli et al., 1994; Milner et al., 1994; Uyttendaele et al., 1996; Weinmaster et al., 1991), which can pair with two serrate like ligands (Jagged 1 & 2) (Lindsell et al., 1995; Luo et al., 1997) or three delta-like-ligands (Dll-1, -3 & -4) (Karanu et al., 2001; Shutter et al., 2000) Notch signaling appears to act at multiple stages of T cell differentiation (Deftos et al., 2000; García-Peydró et al., 2003; Izon et al., 2001; Jiang et al., 1998; Robey et al., 1996; Washburn et al., 1997) The strongest evidence for the role of Notch signaling in T cell development comes from gain-of-function and loss-of-function studies (Allman et al., 2002; Izon et al., 2002; MacDonald et al., 2001; Pear et al., 1996; Pui et al., 1999; Radtke et at, 2002; Wilson et al., 2001), in which signaling though Notch-1 was shown to play a crucial role in determining the B cell versus T cell lineage choice (Pear and Radtke, 2003; Radtke et al., 2002).
HSCs express multiple Notch receptors (Milner et al., 1996; Milner et al., 1994) but the expression patterns of the various Notch ligands have been reported to be distinct between bone marrow stromal cells (Jones et al., 1998; Karanu et al., 2001; Li et al., 1998; Varnum-Finney et al., 1998; Walker et al., 1999) and thymic epithelial cells (Anderson et al., 2001) Taken together, these results suggest that different Notch receptors and ligands may control different aspects of hematopoiesis depending on the microenvironment: allowing for self-renewal in the bone marrow and influencing cell fate decisions in the thymus (Varnum-Finney et al., 1998). This led to the hypothesis that bone marrow stromal lines, such as OP9 cells (Cho et al., 1999; Kim et al., 2003; Kodama et al., 1994), which support B cell differentiation may do so because the appropriate Notch ligand to induce T cell commitment and differentiation is absent. This hypothesis was tested, and demonstrated that OP9 cells, which do not express Dll1, when retrovirally-transduced to express Dll-1 (OP9-DL1) inhibited the development of B cells and favored the development of T cells from fetal liver-derived HSCs (Schmitt and Zúñiga-Pflücker, 2002) or mouse ESCs (Schmitt et al., 2004). Given the high level of homology (90%) between mouse and human Dll-1 molecules, and the observation that mouse stromal lines can support the differentiation of human HSCs (Bennaceur-Griscelli et al., 2001; Jaleco et al., 2001; Karanu et al., 2001; Rawlings et al., 1995), the inventors sought to determine whether human CB-derived HSCs (CD34+CD38−) cultured on OP9-DL1 cells could initiate and support T cell differentiation in vitro.
T-cells develop within the thymus from bone marrow-derived hematopoietic progenitors, and follow a series of stage-specific differentiation events, which are broadly characterized by the developmentally-coordinated expression of CD4 and CD8 (Blom and Spits, 2006; Spits, 2002).
The initial stages of human T-cell development include precursors that express the stem cell marker CD34 (Haddad et al., 2006; Hao et al., 2001), which is also present on hematopoietic stem cells (HSCs) and on multipotent or lineage-specified progenitor cells. Furthermore, several groups have established that the most primitive cells in the human thymus possess multi-lineage potential (Blom et al, 1997; Res et al., 1996; Weerkamp et al., 2006a) as they give rise to T-lineage, as well as, natural killer (NK), dendritic cells (DCs) and to some extent myeloid-lineage cells (Blom et al., 1997; La Motte-Mohs et al., 2007). Within the known hierarchy of T-cell development, the earliest precursor subset is further defined by their lack of CD3, CD4, CD8 and CD1a expression (Galy et al., 1993; Vanhecke et al., 1995).
While immature stages of T-cell development are typically delineated as CD34+CD1a− (most immature) and CD34+CD1a+ cells, these populations remain heterogeneous. Of note, CD7 expression is one of the earliest cell surface markers known to appear during T-lymphopoiesis (Haddad et al., 2006; Haynes et al., 1988). Importantly, the transition from CD34+CD7+CD1a− to CD34+CD7+CD1a+ by early thymocytes is associated with T-cell commitment, as a small percentage (˜10%) of these cells bear rearrangement at the T-cell receptor β-chain (TCRβ) locus (Blom et al., 1999; Dik et al., 2005). In addition, CD34+CD7+CD1a+ cells appear to be T-lineage restricted, as these cells show low precursor activity towards non-T-cell lineages (Spits, 2002). Following this stage, thymocytes progress to a CD4 immature single positive (CD41SP) stage, at which point CD4 is expressed in the absence of CD8. Thereafter, a subset of CD41SP cells are thought to complete TCRβ rearrangement leading to β-selection and differentiation to the CD4+CD8+ double positive (DP) stage. Finally, following TCRα rearrangement, TCRαβ-expressing DP thymocytes undergo positive and negative selection, and yield CD4+CD8− and CD4−CD8+ single positive (SP) T-cells, which emigrate to the periphery (Vanhecke et al., 1997).
Current understanding of the above-outlined stages has been obtained from analyses of human fetal or adult thymocyte subsets, and by analyzing T-cell development in vitro using xenogeneic engraftment of mouse fetal thymus organ cultures (FTOCs) (Fisher et al., 1990; La Motte-Mohs et al., 2007). While these systems have provided important insight into T-cell development, the capacity to evaluate specific progenitor populations has remained difficult to assess given the requirement of human thymus tissue, and the limited number of progenitor T-cells that can be readily analyzed.
Previous work from the inventors' laboratory established that human T-lineage differentiation can be induced from umbilical cord-blood (UCB)-derived HSCs cocultured with OP9-DL1 cells (La Motte-Mohs et al., 2005). The inventors showed normal stage-specific expression of various cell surface molecules, including the generation of immature DP T-lineage cells. However, these studies were not performed using quantitative clonal analyses, and it was unresolved whether different UCB CD34+ subsets could give rise to T-lineage cells and whether Delta-like/Notch signals influence the T-progenitor frequency of CD34+ UCB cells. Additionally, it was unclear whether functional T-cells could be generated. Finally, the inventors' initial studies (La Motte-Mohs et al., 2005) showed that during the early stages of HSC/OP9-DL1 differentiation a population of cells resembling T-progenitors became apparent, however the potential of these cells to serve as effective T-cell progenitors was not addressed.